The longterm objectives of this research proposal are to understand in greater detail the mechanisms of Na+ and C1- absorption and K+ secretion by the urinary bladder of the winter flounder. This epithelium is unique in several aspects. It absorbs Na+ and C1- by a process which can be conducted in the absence of a transepithelial voltage or short-circuit current. Thus, the absorption is an electrically silent process. The bladder is a high resistance epithelia which makes the determination of transcellular fluxes more accurate than is the case for epithelia possessing a lower paracellular resistance. Another interesting feature is that in some bladders there does exist a short-circuit current which is completely attributable to K+ secretion into the lumen. Thus, one is able to determine independently NaC1 absorption and K+ secretion. An additional unique feature of this epithelia is its lack of sensitivity to furosemide and its sensitivity to thiazide diuretics. Thus, this epithelia is the only in vitro model for the mechanism of action of the thiazide diuretics. It appears that these diuretics inhibit a neutral NaC1 cotransporter which, in contrast to the furosemide-sensitive cotransporter, does not require K+ for its operation. The specific aims for the project are: 1) to determine the dose-response relationships for a variety of thiazide-type diuretics using tracer fluxes and electrophysiologic techniques; 2) to determine the mechanism of action of papaverine (which also inhibits NaC1 absorption) using electrophysiologic approaches; 30 to determine the kinetics of NaC1 absorption including the interrelationships between the mucosal concentration of Na+ and C1- and the stoichiometry of the cotransporter; 4) to examine the factors regulating basolateral membrane ion permeation; and 5) to conduct a detailed morphological examination of the bladder, its response to inhibitors of transport, and the pathways of lanthanum permeation. The understanding of this transport system will likely assist us in understanding the operation of the distal convoluted tubule of the kidney, the mechanism of action of thiazide diuretics. The results may lead to important opportunities for localizing the NaC1 cotransporter and determining its biochemical nature.